The mutualistic association between sepiolid squids (Mollusca: Cephalopoda) and their Vibrio symbionts is an experimentally tractable model to study the evolution of animal and bacterial associations at various levels of interaction (genetics, physiology, population ecology). Since symbiotic bacteria are environmentally transmitted to new hosts with every generation, it provides an opportunity to resolve how the ecology of the free-living symbiont affects the architecture of bacterial-host interactions. Bacteria encounter potentially conflicting selective pressures, competing with one another to colonize and persist in the sepiolid light organ, but also competing for scarce resources in the environment outside the squid. A hierarchy of colonization exists, where particular V. fischeri strains out-compete other V. fischeri strains for colonization of their native squid partner, and abiotic factors such as water and temperature can affect the fitness of different strains. Genetic factors that contribute to intra-strain variations in fitness have not been elucidated, and neither have the genetic factors that likely contribute to intra-strain fitness in the environment. The following project will examine how genes that are highly-expressed exclusively in sea water or expressed exclusively in the light organ contribute to bacterial fitness. The present proposal is an extension of the current work in my laboratory, and focuses on four specific goals: i) Characterize environmentally-relevant phenotypes of strains with null mutations in highly-expressed, variable genes. ii) Determine whether a mutant strain complemented with the wild-type operon from the same strain is fit for environmental persistence (free-living) than the same strain complemented with the operon from a different strain. iii) Examine whether symbiotic strains with specific variable loci complemented with the wild-type operon or gene from the same strain are fit for host colonization than the same strain complemented with the operon/gene from a different strain. iv) Measure whether symbiotic genes are found in pathogenicity islands through whole genome comparisons, and demonstrate positive selection of these genes compared to normal housekeeping genes.